Melatonin Increases The Sensitivity Of Hepatocellular Carcinoma To Sorafenib By Inhibiting Endoplasmic Reticulum Stress-Related Autophagy

Background Hepatocellular carcinoma is one of the most common malignant tumors of the digestive system,and its incidence and mortality are on the rise worldwide.The prognosis of hepatocellular carcinoma is poor due to its low early diagnosis rate,high recurrence rate after surgery,and high drug resistance.Sorafenib is currently a clinically used regimen for the treatment of advanced hepatocellular carcinoma,which acts as a multi-target tyrosinase inhibitor to inhibit tumor cell growth and tumor angiogenesis to exert anti-tumor effects.However,only 30% of patients with hepatocellular carcinoma are effective for the treatment of sorafenib,and most of them develop sorafenib resistance in the short term.Endoplasmic reticulum stress refers to the dysfunction of the endoplasmic reticulum due to various pathological factors inside and outside of the cell,resulting in the accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum.In response to endoplasmic reticulum stress,cells activate the UPR pathway in an attempt to correct endoplasmic reticulum stress and restore normal function.Three endoplasmic reticulum transmembrane sensors,include protein kinase RNA-like ER kinase(PERK),inositol requires 1 α(IRE1 α)and activated transcription factor-6(ATF6),which constitute the three major pathways of UPR.However,under long-term stress conditions,endoplasmic reticulum stress and UPR cause cells to directly apoptosis.Autophagy as a conservative catabolic process can maintain normal cellular metabolism and homeostasis by degrading intracellular aging organelles andnon-functional proteins.Autophagic bodies with a bilayer membrane structure envelop the organelles and proteins that need to be degraded and fuse with lysosomes to form autophagosomes to achieve degradation,recovery and recycling of cellular components.Through the process of autophagy,cells can survive for several days or even weeks under a range of pathological stresses.Melatonin is an indoleamine secreted by the pineal gland with various biological functions including anti-oxidant and anti-inflammatory properties,and its anti-tumor effect has been continuously emphasized in recent research.Studies have shown that melatonin can enhance the inhibition of tumor growth by combinating with chemotherapy drugs,and has been verified in renal cell carcinoma,melanoma,and thyroid tumors.However,it is currently poorly understood to melatonin increase the sensitivity of hepatocellular carcinoma(HCC)to sorafenib.Objective(1)To explore the effect of melatonin combined with sorafenib in the treatment of hepatocellular carcinoma.(2)To elucidate the role and association of endoplasmic reticulum stress and autophagy in resistance mechanism to sorafenib.(3)To define the specific mechanism by which melatonin affects the efficacy of sorafenib.Methods(1)Set the different concentration gradient of sorafenib on the hepatocellular carcinoma for 24,48,72 h,MTT assay to detect the cell inhibition rate,and calculate the IC50;set different concentrations of melatonin and different concentrations of sorafenib,the MTT assay was used to detect the cell inhibition rate;Once the concentration of melatonin and sorafenib was determined,the apoptosis rate was determined by flow cytometry and TENEL,the expression of Bax and Bcl-2 was detected by Western Blot.(2)Sorafenib with different concentration gradients and time gradients were setfor hepatocellular carcinoma cells.Western blot was used to detect the expression of endoplasmic reticulum stress-related proteins GRP78,IRE1α,PERK,ATF6,XBP1 S,ATF4 and autophagy-related proteins P62,Beclin1,LC3.Hepatocellular carcinoma cells were pretreated with endoplasmic reticulum stress-specific inhibitor TUDC,PBA and autophagy inhibitor 3-MA,and then treated with sorafenib.The apoptosis rate of each group was observed by flow cytometry.Western Blot assay was used to detect the expression of Bax and Bcl-2.(3)Seventy-two surgically resected hepatocellular carcinoma tissues were collected and microarray.The expression levels and correlation of UPR-related pathway proteins PERK,ATF6,IRE1α and autophagy-related protein Beclin1 were observed by immunohistochemistry.Further clinical and pathological data were obtained and analysis with overall survival.Hepatocellular carcinoma cells were treated with endoplasmic reticulum inhibitors TUDC and PBA,and then the expression of autophagy-related proteins P62,Beclin1,and LC3 was detected by Western Blot;Hepatocellular carcinoma were also treated with autophagy inhibitor3-MA,the expression of endoplasmic reticulum-associated proteins GRP78,PERK,ATF6,IRE1 α and ATF4 was observed by Western Blot.Under the treatment of sorafenib,PBA and 3-MA treated hepatocellular carcinoma respectively,and immunofluorescence detected the expression and localization of GRP78 and LC3.(4)Under the treatment of Sorafenib,hepatocarcinoma cells was transfected with small interfering RNA to block the three pathways of UPR,and the expression of autophagy-related proteins Beclin1,LC3 and P62 were detected by Western Blot.The autophagic vacuoles were observed by electron transmission.acridine oranges staining was used to detect the changes of the number of acidic vesicles.At the same time,small interfering RNA was used to interfere with the downstream protein of the most inhibitory UPR pathway,and the expression of autophagy-related proteins Beclin1,LC3 and P62 was also detected by Western Blot.(5)The synergistic of melatonin and sorafenib was detected,and the expression of autophagy-related proteins Beclin1,LC3,P62 and the expression of UPR pathway proteins related to autophagy were detected by Western Blot.Acridine orangeStaining was used to detect the changes of the number of acidic vesicles.Results(1)The MTT results showed that the IC50 of sorafenib on HepG2 cells 24 h,48h,72 h was 26.847 μM,12.156 μM,and 7.217 μM,respectively.At the same time,MTT assay showed that the melatonin(10-5-10-3mol / L)can significantly increase the inhibition of sorafenib on the proliferation of Hep G2 cells.It is worth noting that low concentrations of melatonin(10-5 mol / L)significantly increased the death of Hep G2 cells while the concentration of sorafenib was 5 μ M and 10 μ M,respectively.Therefore,10-5mol / L and 10 μ M were used as concentrations for subsequent experiments to study the synergistic effect of melatonin and sorafenib.The results of flow cytometry and TUNEL showed that melatonin combined with sorafenib caused a higher apoptosis rate of hepatocarcinoma cells than that of sorafenib alone.Western Blot results showed that the Bcl-2/Bax ratio of the combination group was lower than that of the sorafenib alone.(2)Western Blot results showed that the expression of GRP78,IRE1 α,PERK,ATF6,XBP1 S,ATF4 and Beclin1,LC3 increased and P62 decreased with the concentration and time of sorafenib.Flow cytometry and TUNEL results showed that PBA,TUDC and 3-MA combined with sorafenib significantly increased the apoptotic rate of hepatocarcinoma cells compared with the sorafenib alone group.Western Blot results showed that the Bcl-2/Bax ratio of the combination group was lower than that of the sorafenib alone group.(3)Immunohistochemistry results showed that autophagy-related protein Beclin1 was highly correlated with the expression of UPR pathway protein in human hepatocarcinoma tissue samples,especially with PERK.Moreover,patients with the combined expression of PERK and Beclin1 has higher clinical stages and shorter overall survival time.Western Blot results showed that endoplasmic reticulum stress inhibitors PBA and TUDC significantly inhibited sorafenib-induced autophagy,but autophagy inhibitor 3-MA had no defined effect on sorafenib-induced endoplasmic reticulum stress.Immunofluorescence results showed that PBA not only decreased theexpression of sorafenib-induced GRP78,but also inhibited the expression of LC3.(4)Western Blot results showed that interference with PERK expression reduced the increase of LC3 conversion and the increase expression of Beclin1 and increased the expression of P62 induced by sorafenib compared to IRE1αand ATF6.Electron microscopy showed interfering PERK could significantly reduce the increase of sorafenib-induced autophagic vacuoles;acridine orange staining and flow cytometry results showed that the number of acidic vesicles decreased significantly after PERK interference.Western Blot results showed that the expression of LC3-II,Beclin1 decreased,and P62 expression increased after interference with ATF4 as the downstream of PERK compared with sorafenib alone.Western Blot results showed that interference with ATF4 expression reduced Beclin1 expression in Hep G2,7721,and LO2 cells without the treatment of sorafenib.(5)Western Blot results showed that melatonin can decrease the expression of LC3 II,Beclin1,PERK and ATF4 and increase the expression of P62 compared with sorafenib alone.Both acridine orange staining and flow quantification showed that melatonin can reduce the increase number of sorafenib-induced acid vesicles.Conclusion(1)Low concentration(10-5 mol / L)of melatonin increase the sensitivity of liver cancer cells to sorafenib.(2)Sorafenib induce endoplasmic reticulum stress and autophagy in hepatocellular carcinoma,and inhibition of endoplasmic reticulum stress and autophagy can increase sorafenib-induced apoptosis.(3)In hepatocellular carcinoma tissue samples,the expression of UPR pathway protein PERK and autophagy-related protein Beclin1 was highly correlated,and patients with combined expression of PERK and Beclin1 had a worse prognosis.(4)Sorafenib-induced endoplasmic reticulum stress is associated with autophagy,and endoplasmic reticulum stress trigger autophagy via the PERK-ATF4-Beclin1 pathway.(5)Sorafenib-induced endoplasmic reticulum stress-related autophagy mediates the resistance mechanism of sorafenib,melatonin increases the sensitivity of hepatocellular carcinoma to sorafenib by inhibiting endoplasmic reticulum-associatedautophagy.